Complex proteins are increasingly used in research, diagnostics and therapeutics. Many of these proteins can only be produced in appropriate eucaryotic cells. With the advent of hybridoma technology and other progress in genetic engineering of eucaryotic cells, mammalian or yeast cell lines are becoming the method of choice for producing complex proteins on a large scale.
The secreted product needs to be purified from the cell culture medium. Most mammalian cells require serum which contains a diverse mixture of proteins, many of which are present at high concentrations. Even in serum-free media systems, numerous other proteins are secreted from the cells. For most of the applications the final product has to meet high levels of purity and activity.
The successful production of these proteins depends largely on the development of fast and efficient methods of purification. Typically, the purification constitutes the major cost (up to 80% of the total cost) in these processes. The large scale use of these protein products is hindered because of the high cost.
There is an urgent need for processes to produce proteins in a simple and economical way. Significant cost reduction in the production of protein biologics could be realized if the purification Would be integrated with cell culture into a fully automated system. In addition, the product quality is also expected to improve because the secreted protein is continuously removed from the culture medium in which the product is exposed to catabolic enzymes. Protein identity is an important issue for protein biotherapeutics, i.e. the final product should be free of degraded or other aberrant protein molecules. The integration requires that the presence of the purification unit in the cell culture system would not affect the conditions of cell culture. Therefore, highly-specific purification methods like affinity/immunoaffinity chromatography is needed to make the integration of cell culture and purification feasible for the continuous purification of secreted product.
Progress in cell culture technology has led to the development of membrane bioreactors for growing eucaryotic, such as mammalian cells within well-defined compartments. Cells grown inside low nonspecific adsorption flat sheet or hollow fiber membranes in thin (200-400 .mu.m) layers are continuously perfused with nutrients and grow to cell densities previously unattainable by the stationary, stirred tank or airlift-type fermentors. Nutrient deprivation or shear sensitivity issues are minimized by this technology. This allows high cell viability in the bioreactor and minimize DNA contamination of the product. The microfiltration membrane eliminates the opportunity of bacterial contamination of the bioreactor. The cells are grown at tissue density with high production rates surpassing the production capacity of conventional bioreactors. After populating the available compartment space, the cells reach a growth-arrested state in which most of their energy is directed towards production. This configuration allows the highest production capacity per unit volume of bioreactor space.
Another important aspect of the integration is the availability of appropriate protein separation technologies. Current protein purification technologies require significant improvement in order to realize the potentials of the integration concept. A major obstacle is that the interaction of the cell culture medium with the protein separation material (chromatography resin) may change the composition of the medium which can be detrimental to the cells in culture. Chromatography media like ion exchange or hydrophobic matrices can drastically change the culture medium composition and thus are unsuitable for an integrated instrument if continuous removal of the product is desired. Biospecific, affinity separation is the only method offering the least interference with cell culture. However, current affinity technologies have serious shortcomings which have prevented them from being incorporated into an integrated system.
The integration of cell culture with continuous purification of secreted product without jeopardizing the cell culture by introducing potentially toxic chemicals and bacterial/vital contamination necessitates the development of a stable, nontoxic, chemically inert, sterilizable activated affinity chromatography resin. Current activated affinity matrices cannot be incorporated into the integrated instrument because they do not meet the criteria of being chemically inert, nontoxic, stable, and sterilizable. The most commonly used coupling methods employ reactive electrophilic centers with leaving group displaced by the incoming nucleophilic ligand (protein/antibody). These displacement reactions frequently remain incomplete even after capping the unreacted sites and continue to release leaving groups, many of them are toxic to cells. Conversely, constituents of the culture medium may be covalently attached to the matrix. The affinity resin may leach other toxic molecules, like isocyanate from CNBr-activated matrices, for long periods of time. This is toxic to the cells in the bioreactor. The iramobilization method may also increase the protease sensitivity of immobilized protein (antibody) ligand, an issue which is a problem with the traditional coupling chemistries.
In affinity separations, proteins (antibodies) are frequently used as ligand. In the integrated system, the immunoaffinity chromatography resin must withstand the conditions of cell culture for long periods of time. The warm, highly-oxygenated environment of cell culture medium may diminish the activity of the immunoaffinity column. Many of the cultured mammalian cells, including hybridomas, secrete proteolytic enzymes which may degrade the immobilized antibody ligand. The same applies to dead cells spilling their content into the culture medium. The structure of the support and the method of immobilization also plays an important role in the protease sensitivity of immobilized antibody. The low concentration of secreted proteins in the cell culture medium may also complicate quantitative recovery of the product.
Current immunoaffinity technologies make process automation complicated because of the continuous loss of immunosorbent capacity. This is the result of ligand leaching and inactivation of immobilized antibody for reasons mentioned above. The total cycle life of the immunoadsorbent (5-30 cycles) is usually too short to make this technology suitable for the integration of bioreactor and purification for continuous product recovery. All these issues need to be addressed in order to make affinity chromatography media compatible with mammalian cell culture.